Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes
One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kit...
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| Published in | ACS Omega Vol. 6; no. 11; pp. 7374 - 7386 |
|---|---|
| Main Authors | , , , , , , , , , , , , , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
United States
American Chemical Society
23.03.2021
American Chemical Society (ACS) |
| Subjects | |
| Online Access | Get full text |
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| Abstract | One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and Thermus aquaticus DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility. |
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| AbstractList | One-step reverse-transcription
quantitative polymerase chain reaction
(qRT-PCR) is the most widely applied method for COVID-19 diagnostics.
Notwithstanding the facts that one-step qRT-PCR is well suited for
the diagnosis of COVID-19 and that there are many commercially available
one-step qRT-PCR kits in the market, their high cost and unavailability
due to airport closures and shipment restriction became a major bottleneck
that had driven the desire to produce the key components of such kits
locally. Here, we provide a simple, economical, and powerful one-step
qRT-PCR kit based on patent-free, specifically tailored versions of
Moloney murine leukemia virus reverse transcriptase and
Thermus aquaticus
DNA polymerase and termed R3T (Rapid
Research Response Team) one-step qRT-PCR. We also demonstrate the
robustness of our enzyme production strategies and provide the optimal
reaction conditions for their efficient augmentation in a one-step
approach. Our kit was routinely able to reliably detect as low as
10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our
kit successfully detected COVID-19 in clinical samples of broad viral
titers with similar reliability and selectivity to that of the Invitrogen
SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR
kits. Overall, our kit has shown robust performance in both laboratory
settings and the Saudi Ministry of Health-approved testing facility. One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and Thermus aquaticus DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility.One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and Thermus aquaticus DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility. One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility. One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and Thermus aquaticus DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility. |
| Author | Takahashi, Etsuko Takahashi, Masateru Alsomali, Afrah Pain, Arnab Almontashiri, Naif A. M Lee, Jae Man Salunke, Rahul Hala, Sharif Alhamlan, Fatimah S Hashem, Anwar M Hamdan, Samir M Mfarrej, Sara Kusakabe, Takahiro Al-Qahtani, Ahmed A Ramos-Mandujano, Gerardo Mon, Hiroaki Tehseen, Muhammad Sakashita, Kosuke Alofi, Fadwa S Sobhy, Mohamed A Khogeer, Asim Li, Mo |
| AuthorAffiliation | Vaccines and Immunotherapy Unit, King Fahd Medical Research Center Laboratory of DNA Replication and Recombination, Biological and Environmental Sciences and Engineering Division King Abdullah International Medical Research Centre, Jeddah, Makkah Laboratory of Insect Genome Science Ministry of National Guard Health Affairs Department of Infection and Immunity Taibah University Plan and Research Department King Fahad Hospital Center for Genetics and Inherited Diseases Department of Infection and Immunity, King Faisal Specialist Hospital and Research Centre Pathogen Genomics Laboratory, BESE Division Infectious Diseases Department General Directorate of Health Affairs Makkah Region, MOH King Abdullah Medical Complex (KAMC) Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences Department of Medical Microbiology and Parasitology, Faculty of Medicine King Saud Bin Abdulaziz University of Health Sciences Stem Cell and Regenration Laboratory. Biological and Environmental Sciences and E |
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| Cites_doi | 10.1099/0022-1317-79-12-2921 10.1093/nar/20.7.1487 10.1016/j.chroma.2020.461051 10.1016/j.virusres.2006.01.017 10.1126/science.1118391 10.1038/s41586-020-2008-3 10.1093/clinchem/hvaa102 10.1128/mmbr.69.4.635-664.2005 10.1016/j.jcv.2020.104412 10.1038/s41586-020-2012-7 10.1038/s41596-019-0210-2 10.1007/s12079-008-0026-2 10.1128/jb.127.3.1550-1557.1976 10.1016/j.cmi.2020.04.001 10.1016/s0021-9258(17)39369-9 10.1016/j.tibtech.2011.01.007 10.1128/jvi.01358-06 10.1128/jvi.15.4.785-797.1975 10.1016/j.virusres.2020.198129 10.1038/2261209a0 10.1016/s0166-0934(03)00067-3 10.1371/journal.ppat.1006698 10.1016/j.aspen.2019.02.008 10.1038/s41587-020-0513-4 10.1016/j.ab.2006.10.009 10.2807/1560-7917.es.2020.25.3.2000045 10.1016/j.jcv.2009.06.001 10.1038/s41586-020-2202-3 10.1016/0003-2697(90)90238-5 10.1016/s0140-6736(20)30251-8 10.1038/s41579-018-0118-9 10.1016/j.puhe.2020.04.009 10.1128/jvi.15.4.843-854.1975 10.1016/s0140-6736(20)30154-9 10.1038/nature12711 10.1016/j.jmoldx.2020.03.006 10.1016/j.cub.2020.05.023 |
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| Contributor | Department of Infection and Immunity, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia King Abdullah International Medical Research Centre, Jeddah, Makkah, Ministry of National Guard Health Affairs, Jeddah, Makkah 22384, Saudi Arabia King Abdullah Medical Complex (KAMC), Jeddah 23816, Saudi Arabia College of Applied Medical Sciences, Taibah University, Madinah 41311, Saudi Arabia Bioscience Program Proteomics, protein expression & cytomet Department of Medical Microbiology and Parasitology, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan King Saud Bin Abdulaziz University of Health Sciences, Jeddah 22384, Saudi Arabia Infectious Diseases Department, King Fahad Hospital, Madinah 3177, Saudi Arabia Center for Genetics and Inherited Diseases, Taibah University, Madinah 42353, Saud |
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| Copyright | 2021 The
Authors. Published by American
Chemical Society 2021 The Authors. Published by American Chemical Society. 2021 The Authors. Published by American Chemical Society 2021 The Authors |
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| Snippet | One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the... One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the... |
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| SubjectTerms | 3T Biochemistry Biophysics Biotechnology Chemistry Computational Biology COVID -19 COVID -19 Diagnostics Developmental Biology enzyme production strategies Genetics Health-approved testing facility Immunology In-House Enzymes One-step reverse-t Infectious Diseases Invitrogen SuperScript III Platinum Molecular Biology Moloney murine leukemia virus One-Step qRT-PCR Kit polymerase chain reaction QD1-999 Rapid Research Response Team RNA Thermus aquaticus DNA polymerase Virology |
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| Title | Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes |
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